intro to chromatography

INTRODUCTION TO
CHROMATOGRAPHY

Pusat Pengajian Sains Kimia & Teknologi Makanan, 1
Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.

What is Chromatography?

Pusat Pengajian Sains Kimia & Teknologi Makanan,

Solvent extraction:
Extraction is the transfer of a solute from one phase to
another

Phase 2

Phase 1

S(in phase 1) S(in phase 2)

Extraction of Uranyl Nitrate from water into
[S]2
ether Partition coefficient: K ≈
[S]1
Chromatography:
Chromatography operates on the same principle as extraction,
but one phase is held in place while the other moves past it.

Chromatography:
Chromatography operates on the same principle as
extraction, but one phase is held in place while the other
moves past it.
Air flow



Air flow


Fresh solvent
(eluent)

Initial band
A and B solutes A

Column packing
(stationary phase) B
Suspended in solvent
(mobile phase)

Solvent flowing out A emerges
(eluate)
B emerges



It is the collective term for a family of laboratory
techniques for the separation of mixtures by distribution
of its components between a mobile and stationary phase
over time
Mobile phase: Stationary phase:
solvent moving through substance which is fixed in
the plate or column place for the chromatographic
(liquid or gas) procedure

The partitioning of solutes between the mobile and
stationary phases gives rise to separation

All chromatographic separations depend on the reversible
sorption and desorption of the components of the mixture in
the stationary and mobile phase

Chromatographic principle
Mobile phase

Equilibrium
Sample established
mixture at each point
(ideally)
Stationary phase

The molecules of the Retardation of
mixture interact with rate of
the molecules of the movement of
Mobile and Stationary molecules
Phase
Different distribution
Each molecule interacts
coefficients and different
differently with MP and SP
net rates of migration

Purpose of Chromatography
Analytical Chromatography – separate and
identify or measure the relative proportions of
components in a mixture
300 L
Preparative Chromatography - purify and
collect one or more components of a sample for
further use
The two are not mutually exclusive

Scaling Up:
2
Large mass Large column radius
=
Small mass Small column radius Purifies 1 Kg of material


Types of Chromatography

Chromatography is divided into
categories on the basis of the mechanism
of interaction of the solute with the
stationary phase


Classification based on the chromatographic principle
Stationary Type of
Mobile Phase
Stationary Phase Phase Support Chromatography Acronym

Gas Column Gas-Solid Chromatography GC/GSC
Adsorption
Liquid column Chromatography
Chromatography LC
Column High Performance Liquid
Competition between a solid HPLC
Liquid Chromatography
adsorbent and the mobile
phase Thin Layer Chromatography TLC
Planar layer
Paper Chromatography PC
Gas-Liquid Chromatography
Partition Gas Column Supercritical Fluid
GC/GLC
SFC
Chromatography Chromatography
Competition between a liquid
Liquid-Liquid Chromatography
stationary phase and the
Liquid Column High Performance Liquid LC/HPLC
mobile phase
Chromatography

Ion Exchange
Chromatography Ion-Exchange Chromatography
IEC
Competition between an ionic Liquid Column High Performance Ion
IC/HPIC
exchange resin stationary Chromatography
phase and liquid mobile phase

Permeation
Chromatography
Competition between a Liquid Column Gel Permeation Chromatography GPC
polymer matrix stationary
phase and liquidPusat Pengajian Sains Kimia & Teknologi Makanan,
mobile phase

Classification of Chromatography

Based on Stationary Phase Support
Planar: Stationary Phase supported on a flat plate
or in pores of paper. Mobile Phase moves through
Stationary Phase by capillary action, gravity or
electrosatic forces

Column: Stationary Phase held in a tube and
Mobile Phase forced through


Classification according to the phases

Chromatography

Mobile Phase = Liquid Mobile Phase = Gas
Liquid Chromatography Gas Chromatography

Stationary Stationary Stationary Stationary
Phase = Solid Phase = Liquid Phase = Solid Phase = Liquid
LSC LLC GSC GLC

Partition of Partition of
Adsorption or ion analytes analytes
Adsorption
exchange between two between two
phases phases


Classification according to physical features of the
analytes

Molecular
Physical property Separation technique
characteristic

volatility GLC
Polarity solubility LLC
adsorptivity LSC

Ion Exchange Chrom.
Ionic charge
Electrophoresis

Gel Permeation Chrom.
diffusion
Size (mass) Dialysis

sedimentation Ultracentrifugation
Shape ligand binding Affinity Chrom.


Methods based on Ionic Nature of the sample

Ion nature of
the sample is
•Number & nature of ionisable groups
affected by: •pH environment
•Presence of other ions
And results
in:
Association of ions Movement in an electric
with opposite charge field

Ion-exchange Electrophoresis
chromatography Capillary electrophoresis

Direction & velocity of
Competition for ionic movement depends on
binding sites on a resin the sign & intensity of
the ionic charge

Methods based on polarity (i.e. affinity of like-molecules
for each other)
Partition between two phases:

Solid/Liquid Liquid/Liquid Liquid/Vapour

A major factor in separation is:

Adsorption Solubility Solubility

And the methods involve:
Solid Two immiscible A solution
adsorbents liquids and its vapour
Methods are generally
known as:
Adsorption Liquid Gas-Liquid
Chromatography Chromatography Chromatography


Methods based on size
Size affects the rate of:

Diffusion Sedimentation
Movement due to
this may be:
Restricted by Balanced by Reduced by high Maximal in low
pore size each other density solvents density solvents
Isodensity Velocity
Diffusion
Fixed pore Variable pore
Ultracentrifugation
size size
Dialysis Gel permeation
chromatography

Gel filtration
Molecular exclusion (largest molecules come out
first as not held by pores)

intro to chromatography

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